MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors

doi:10.1073/pnas.0611192104

MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors

*Gonda Diabetes Center,
^†Division of Molecular Biology,
^‡Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010

Communicated by Arthur D. Riggs, Beckman Research Institute of the City of Hope, Duarte, CA, December 18, 2006 (received for review November 30, 2006)

Abstract

Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-α 1 and -2 (Col1a1 and -2). Transforming growth factor β1 (TGF-β), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-β increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, δEF1. TGF-β treatment or short hairpin RNAs targeting δEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-β also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to δEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-β in MMC. TGF-β treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3′ UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with δEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-β and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-β-induced Col1a2 expression by down-regulating E-box repressors.

Footnotes

^§To whom correspondence should be addressed at:
Gonda Diabetes Center, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010.
E-mail: rnatarajan{at}coh.org
Author contributions: M.K. and R.N. designed research; M.K., J.Z., M.W., L.L., and H.Y. performed research; M.K., J.Z., M.W., and J.J.R. contributed new reagents/analytic tools; M.K., J.Z., J.J.R., and R.N. analyzed data; and M.K. and R.N. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0611192104/DC1.
Abbreviations:

DN,

diabetic nephropathy;

MC,

mesangial cells;

MMC,

mouse MC;

SIP1,

Smad-interacting protein 1;

miR,

microRNA;

ECM,

extracellular matrix;

Col1a1 and -2,

collagen 1-α 1 and 2;

shRNA,

short hairpin RNA;

qPCR,

quantitative PCR;

AS,

antisense;

STZ,

streptozotocin.